Annals of Botany 86: 745752, 2000 doi:10.1006/anbo.2000.1247, available online at http://www.idealibrary.

com on

Organogenesis, Dierentiation and Histolocalization of Alkaloids in Cultured Tissues and Organs of Duboisia myoporoides R. Br.
N U R U S S A B A K H A N A M *{, C H E A N G K H O O {, RO B E R T C LO S E { and A B DU L G . K HA N { {Department of Biological Sciences, University of Western Sydney, Macarthur, PO Box 555, Campbelltown, NSW 2560, Australia and {Department of Chemistry, University of Western Sydney, Macarthur, PO Box 555, Campbelltown, NSW 2560, Australia
Received: 15 November 1999 Returned for revision: 4 February 2000 Accepted: 14 June 2000 Published electronically: 29 August 2000

Duboisia myoporoides R. Br. shoots were regenerated from non-organogenic and organogenic calli induced with nine dierent cytokinin/auxin combinations. Alkaloid colour reagents localized tropane alkaloids in the vascular regions which had large cells in the secondary xylem of the basal stem sections of the non-rooted shoots. Tropane alkaloids were localized in shoots regenerated from calli induced with two dierent cytokinin/auxin combinations. No alkaloids were localized in shoots regenerated from calli induced with other cytokinin/auxin combinations. However, only nicotine was detected by GC-MS in the non-rooted shoots regenerated from calli induced with three dierent cytokinin/auxin combinations. Tropane alkaloids were also localized in xylem cells of roots regenerated from calli induced with two dierent cytokinin and auxin combinations independently. The presence or absence of nicotine, hyoscyamine and scopolamine in dierent cultured plant materials was conrmed by GC-MS, indicating that although the root is the main site for alkaloid biosynthesis, with suitable cell dierentiation, alkaloid biosynthesis # 2000 Annals of Botany Company may take place in cultured shoots without root initiation. Key words: Duboisia myoporoides, Corkwood tree, Solanaceae, tropane alkaloid, alkaloid localization, shoot culture, root culture, iodoplatinate.

I N T RO D U C T I O N Duboisia myoporoides R. Br., an Australian member of the Solanaceae family, contains dierent groups of alkaloids and is cultivated in Australia for its high scopolamine content (Endo and Yamada, 1985; Grin, 1985; Gritsanapan and Grin, 1991). The tropane alkaloids hyoscyamine and scopolamine are medicinally important (Evans, 1990; Hashimoto and Yamada, 1992). In the complete D. myoporoides plant, alkaloid biosynthesis takes place in the root cells (Hashimoto and Yamada, 1992). While dierent classes of alkaloids have been detected in cultured roots of this species (Endo and Yamada, 1985; Yukimune et al., 1994), tropane alkaloids are found in the cultured shoot only after root initiation (Bhandary et al., 1969; Lin and Grin, 1992a). D. myoporoides shoots were regenerated from nonorganogenic and organogenic calli induced with dierent cytokinin and auxin combinations. Non-organogenic calli occur when the mass of callus increases, but no shoot-buds or roots are formed. These non-organogenic calli require dierent cytokinin/auxin combinations at dierent stages to regenerate shoots from the calli (Kitamura et al., 1980). Organogenic calli, in contrast, appear to require one cytokinin and auxin combination for all stages of shoot regeneration (Kukreja et al., 1986). These authors did not report the alkaloid biosynthetic ability of non-rooted shoots.
* For correspondence. Fax 02 4620 3793, e-mail dk.williams@uws. edu.au

Alkaloid colour reagents have been used to localize tropane alkaloids in Atropa belladonna (James, 1950) and Erythroxylum coca and E. novogranatense (Ferreira et al., 1998), but not in D. myoporoides. This work investigates alkaloid localization and the production of nicotine, hyoscyamine and scopolamine in dierent stages of shoot regeneration from calli induced with dierent cytokinin/auxin combinations. Alkaloid localization and nicotine, hyoscyamine and scopolamine production are compared in cultured non-rooted shoots and cultured roots without aerial organs. M AT E R I A L S A N D M E T H O D S All tissue culture experiments were carried out in duplicate with 20 replicates per experiment. All cultures were transferred to fresh media at intervals of four to ve weeks and incubated at 25 + 28C. For light incubation, a 14 h photoperiod of cool-white light was provided by a 35 W (Osram) uorescent tube with a photon irradiance of 15.2 mmol quanta m 2 s 1 (PAR) at a distance of 40 cm from the light source. Plant materials and callus induction Material of Duboisia myoporoides R. Br. was collected from a mature tree growing in the Mount Annan Botanic Gardens, Campbelltown NSW, Australia. Fresh, healthy and mature leaves collected from the tree were surface sterilized by placing them in 70 % v/v ethanol for 30 s # 2000 Annals of Botany Company

0305-7364/00/100745+08 $35.00/00

746 Khanam et al.Alkaloid Localization in D. myoporoides R. Br. Cultured Tissues and Organs
followed by two treatments of 10 min in 1 % hypochlorite solution and three washes with sterile water. Twenty 1 cm2 surface-sterilized leaf segments were placed separately on MS (Murashige and Skoog, 1962) basal medium adjusted to pH 5.7 and supplemented with 3 % sucrose, 0.9 % agar and the following cytokinin/auxin combinations: (1) 2,4-dichlorophenoxy acetic acid (2,4-D) 0.1 mM; (2) benzyladenine (BA) 10 mM naphthalene acetic acid (NAA) 1 mM; (3) BA 10 mM 2,4-D 0.1 mM; (4) 6-furfuryl amino purine (kinetin or Kin) 10 mM indolyl-3-acetic acid (IAA) 1 mM; (5) Kin 10 mM indolyl-3-butyric acid (IBA) 10 mM; (6) Kin 10 mM NAA 10 mM; (7) Kin 10 mM 2,4-D 0.1 mM; (8) BA 10 mM IAA 1 mM; (9) BA 10 mM IBA 0.1 mM; and (10) BA 1 mM IBA 10 mM independently. [These supplements are referred to as combinations (1) to (10) in the text.] The cultures were incubated in the dark. Non-organogenic calli induced with combinations (1)(7) were incubated in their respective media in the dark for 12 weeks ( from the date of callus induction). The 11-week-old calli (callus induction was started after 1 week) were used for alkaloid localization and for chemical analysis and shoot culture. After 4 weeks, small organogenic calli induced with combinations (8) and (9) were incubated in the light for shoot-bud induction. The small organogenic calli induced with combination (10) were incubated in the dark. Shoot culture from organogenic calli Small calli induced with combinations (8) and (9) were incubated in their respective media. Shoot-bud induction and shoot elongation were carried out in the medium supplemented with the same cytokinin and auxin combinations used for callus induction. The cultures were incubated in the light for 18 weeks after callus induction. Two-weekold calli, 14-week-old calli with dierentiated shoots, and the basal stems of 9-week-old non-rooted shoots were examined for localization of alkaloids. Two-week-old calli, 14-week-old calli without dierentiated shoots, 3-week-old shoot-buds and 4- and 9-week-old dierentiated non-rooted shoots were analysed by GC-MS. Root culture from non-organogenic calli Eleven-week-old calli induced with combination (2) were transferred to a medium supplemented with full MS salt full MS vitamin IBA 25 mM and incubated for 6 weeks. After formation of roots, cultures were incubated for another 4 weeks. Root culture from organogenic calli Calli induced with combination (10) were incubated in the basal medium supplemented with the same cytokinin and auxin combinations for root induction and elongation. After formation of roots, the cultures were incubated for a further 4 weeks. All root cultures were incubated in the dark. The 4-weekold roots without aerial organs regenerated from nonorganogenic and organogenic calli were examined for localization of alkaloids and analysed by GC-MS. Statistical analysis Nicotine, hyoscyamine and scopolamine contents of non-rooted shoots regenerated from calli induced with combination (9) and those in roots regenerated from calli induced with combination (10) were compared using a one-tailed two sample t-test. Histolocalization of alkaloids Cultured tissues and organs were collected from ve replicate culture vials and 50 to 60 sections were prepared. The results are presented as the average response of 30 randomly selected sections. All alkaloid stains were matched by controls. Fresh sections were kept in alcoholic tartaric acid solution for 45 weeks to free the sections from alkaloids (Johansen, 1940). The sections were then washed thoroughly three times with fresh water and stained with the alkaloid colour reagents. Free-hand fresh and alkaloid-free sections were stained with iodoplatinate (Stevens, 1986) and platinic chloride (5 % aqueous solution) (Cromwell, 1955) alkaloid colour reagents. (The colour reagents were selected on the basis of their staining results observed on free-hand fresh and

Shoot culture from non-organogenic calli The following steps were carried out to regenerate shoots from calli induced with combinations (1)(7) (see Lin and Grin, 1992b). (a) Production of green calli. Eleven-week-old white/light brown calli induced with seven dierent combinations were transferred independently to the basal medium supplemented with BA 0.1 mM NAA 27 mM and incubated in the light for 2 weeks. The green calli produced were used for chemical analysis and shoot-bud induction. (b) Shoot-bud induction. Green calli developed from 11-week-old white/light brown calli induced with combinations (1)(7) were transferred independently to the basal medium supplemented with BA 22 mM and incubated in the light for 8 weeks. Induced shoot-buds were incubated for 1 more week. The 12 week-old shoot-buds were used for chemical analysis and shoot elongation. (c) Shoot elongation. Shoot-buds were separated from calli and transferred independently to basal medium supplemented with BA 5 mM NAA 0.5 mM and incubated in the dark for 2 weeks. Elongated shoots were then incubated in the latter supplemented medium in the light for 4 weeks. Basal stems of the 6-week-old non-rooted shoots were examined for localization of alkaloids, and shoots with leaves were analysed by GC-MS.

Khanam et al.Alkaloid Localization in D. myoporoides R. Br. Cultured Tissues and Organs 747
alkaloid-free sections of mature plant organs. The two reagents stained cell contents in the vascular region of the stem and xylem cells of the root sections indicating alkaloid localization; unpubl. res.) Three to ve sections were transferred directly to 1 ml of the reagents in a watch glass and were stained for 510 min. Some of the stained sections were de-stained by washing with water. For comparison, small amounts of commercial atropine, scopolamine and nicotine were placed separately on microscope slides and a few millilitres of the staining reagents added individually. The colour produced on the slide was then photographed. The colour produced in the fresh sections was compared with the commercial sample under the microscope as well as after photography. All sections were examined under an inverted microscope tted with a 35 mm camera and photographed at dierent magnications. Alkaloid analysis The analytical method described here was validated by testing for precision, accuracy, recovery, linearity, limits of detection, limit of quantication and sample and standard stability (Nou, 1997). About 0.10.5 g of the air dried sample was sand-ground and 1 ml 10 ppm homatropaine (internal standard) and 10 ml 0.5 M HCl added. The resultant solution was then ltered and the ltrate made alkaline with ammonia solution and passed through an `Extrelut 20' (Merck, Germany) column which was then eluted with 40 ml of dichloromethane : isopropanol (4 : 1 v/v). It was necessary to repeat the whole procedure twofour times to obtain a combined extract which was evaporated to dryness and made up with 1 ml methanol for GC-MS analysis using a SGE BPX5 (30 m 22 mm, 0.25 mm lm thickness) column set at 858C for 3 min, rising to 2808C at 128C min 1. The injector and detector temperatures were set at 2208C and 2808C respectively and helium was used as the carrier gas. The mass spectrometer was operated on the electron impact mode. Quantication was achieved by comparing the total ion chromatograms of standard and sample. Amounts of alkaloids were calculated from four replicates. The following typical mass spectra were obtained for the standard nicotine: 162 [M ] (14), 84 (100), 161 (15), 133 (29), 92 (6); hyoscyamine: 289 [M ] (7), 124 (100), 140 (8), 82 (25), 67 (11); and scopolamine: 303 [M ] (21), 94 (100), 154 (30), 138 (74), 108 (47), and those of the sample nicotine: 162 [M ] (15), 84 (100), 161 (13), 133 (25), 92 (6); hyoscyamine: 289 [M ] (7), 124 (100), 140 (13), 82 (25), 67 (12); and scopolamine: 303 [M ] (21), 94 (100), 154 (32), 138 (67), 108 (51).

R E S ULT S Histolocalization of alkaloids in cultured plant materials Meristemoid regions were found in calli induced with growth regulator combinations (1)(7), and shoot-bud meristems were found in calli induced with combinations (8) and (9). No dierentiated cells were found and no alkaloids were localized in the calli. Alkaloids were localized in the shoots dierentiated from calli induced with combinations (2) and (9) (Table 1, Figs 1 and 3). Cells in the alkaloid-free sections remained colourless (Fig. 2). Vascular regions with large cells in the secondary xylem were found in these sections (Fig. 2). No alkaloids were localized in the vascular regions of the shoots dierentiated from calli induced with combinations (1) and (3)(8) (Table 1, Figs 46). Only small cells in the vascular region were found in these sections (Figs 46). Most cells in the 14-week-old calli induced with combination (9) were arranged in a concentric ring. Alkaloids were localized in these cells (Fig. 7). Alkaloid-free sections remained colourless (Fig. 8). No alkaloids were localized in the 14-week-old calli induced with combination (8). Fewer xylem cells were found in roots regenerated from calli induced with combinations (2) and (10) than in the mature plant root. Alkaloid colour reagents localized alkaloids in the xylem cells of the fresh sections (Fig. 9). In the alkaloid-free sections, the xylem cells remained colourless (Fig. 10).

T A B L E 1. Histolocalization of alkaloids in the basal stem sections of cultured non-rooted shoot of D. myoporoides
Alkaloid localization Cytokinin/auxin used for calli induction (1) (2) (3) (4) (5) (6) (7) (8) (9) 2,4-D 0.1 mM BA 10 mM NAA 1 mM BA 10 mM 2,4-D 0.1 mM Kin 10 mM IAA 1 mM Kin 10 mM IBA 10 mM Kin 10 mM NAA 10 mM Kin 10 mM 2,4-D 0.1 mM BA 10 mM IAA 1 mM BA 10 mM IBA 0.1 mM Cells in the vascular region Small cell Large and small cells Small cell Small cell Small cell Small cell Small cell Small cell Large and small cells Fresh section NL L NL NL NL NL NL NL L Alkaloid-free section NL NL NL NL NL NL NL NL NL

L, Localized; NL, not localized.

748 Khanam et al.Alkaloid Localization in D. myoporoides R. Br. Cultured Tissues and Organs

Khanam et al.Alkaloid Localization in D. myoporoides R. Br. Cultured Tissues and Organs 749
T A B L E 2. Alkaloid contents of cultured D. myoporoides organs
Alkaloid contents (calculated from four replicates) (mg g 1 d. wt + absolute s.d.) Nicotine 1018.0 + 1.0 8.5 + 0.2 880.0 + 70.0 ND ND ND 660.0 + 50.0 ND 5.8 + 0.1 86.0 + 4.0 91.0 + 3.0 Hyoscyamine ND 7.8 + 0.1 ND ND ND ND ND ND 38.5 + 0.4 69.0 + 1.0 82.0 + 3.0 Scopolamine ND 6.5 + 0.4 ND ND ND ND ND ND 3.6 + 0.1 37.3 + 0.3 48.2 + 0.6

Cultured organs Shoot (non-rooted)

Cytokinin/auxin used for calli induction (1) (2) (3) (4) (5) (6) (7) (8) (9) 2,4-D 0.1 mM BA 10 mM NAA 1 mM BA 10 mM 2,4-D 0.1 mM Kin 10 mM IAA 1 mM Kin 10 mM IBA 10 mM Kin 10 mM NAA 10 mM Kin 10 mM 2,4-D 0.1 mM BA 10 mM IAA 1 mM BA 10 mM IBA 0.1 mM

Root (without aerial organ) ND, Not detected.

(2) BA 10 mM NAA 1 mM (10) BA 1 mM IBA 10 mM

Alkaloid contents of cultured plant materials Nicotine, hyoscyamine and scopolamine were absent in 11-week-old calli induced with combinations (1) and (3)(7). Nicotine, hyoscyamine and scopolamine contents of the 11-week-old calli induced with combination (2) were 0.62 + 0.01, 0.41 + 0.03 and 0.23 + 0.02 mg g 1 d. wt respectively. Alkaloids were absent in 2-week-old calli induced with combinations (8) and (9) and also in the 2and 3-week-old shoot-buds induced on the non-organogenic and organogenic calli. No alkaloids were detected in the 4-week-old non-rooted shoots dierentiated from calli induced with combinations (8) and (9). Nicotine, hyoscyamine and scopolamine were detected in the shoots dierentiated from calli induced with combinations (2) and (9) (Table 2). Only nicotine was detected in the shoots dierentiated from calli induced with combinations (1), (3) and (7) (Table 2). Alkaloids were absent in shoots dierentiated from calli induced with the other cytokinin and auxin combinations tested (Table 2). Only hyoscyamine (6.9 + 0.2 mg g 1 d. wt) was detected in the 14-week-old calli collected from the base of the shoot dierentiated from calli induced with combination (9). Alkaloids were absent in 14-week-old calli induced with combination (8). Nicotine, hyoscyamine and scopolamine were detected in 4-week-old roots regenerated from calli induced with combinations (2) and (10) (Table 2). Signicantly higher amounts (P 5 0.001) of nicotine, hyoscyamine and scopolamine were produced in the

4-week-old roots regenerated from calli induced with combination (10) than in the dierentiated non-rooted shoots regenerated from calli induced with combination (9).

DISCUSSION The occurrence of a meristemoid region only in calli induced with combinations (1)(7) and a shoot-bud meristem in calli induced with combination (8) and (9) indicates that the cytokinin/auxin combinations used for callus induction were not optimal for cell dierentiation in calli. The presence of small amounts of nicotine, hyoscyamine and scopolamine in the calli induced with combination (2) showed that some cytokinin/auxin combinations can cause alkaloid biosynthesis in calli cells. As the alkaloids were not localized by colour reagents, it appears that their amounts were not sucient to be identiable by the alkaloid colour reagents used. Sipply and Friedrich (1975) reported small amounts of tropane alkaloids in 24-week-old calli cells of D. myoporoides. No alkaloids were detected in shoot-buds induced on non-organogenic and organogenic calli. Yamada and Endo (1984) reported the absence of alkaloids in D. leichhardtii shoot-buds. Lin and Grin (1992a) also reported the absence of alkaloids in Duboisia hybrid shoot-buds. This indicates that tropane alkaloid biosynthesis in Duboisia needs a more advanced stage of dierentiation than that present in the shoot-buds.

F I G S 16. Cross-sections of the basal stems from non-rooted shoots dierentiated from calli induced with a range of growth regulators. Fig. 1. BA 10 mM IBA 0.1 mM, fresh section showing the presence of stained xylem cell contents in the vascular region which indicates alkaloid localization (iodoplatinate). Fig. 2. BA 10 mM IBA 0.1 mM, alkaloid-free section showing large cells in the secondary xylem. The absence of stained xylem cell contents in the vascular region indicates no alkaloid localization (iodoplatinate). Fig. 3. BA 10 mM IBA 0.1 mM, fresh section showing the presence of stained xylem cell contents in the vascular region indicating alkaloid localization ( platinic chloride). Fig. 4. 2,4-D 0.1 mM, fresh section showing the vascular region consisting of small cells. Absence of stained xylem cell contents indicates no alkaloid localization (iodoplatinate). Fig. 5. Kin 10 mM IAA 1 mM, fresh section showing vascular region with small cells, absence of stained xylem cell contents indicates no alkaloid localization (iodoplatinate). Fig. 6. BA 10 mM IAA 1 mM, fresh section showing vascular region consisting of small cells. Absence of stained xylem cell contents indicates no alkaloid localization (iodoplatinate). Bars 100 mm. xy, Xylem cells; vr, vascular region; lx, large xylem cells.

750 Khanam et al.Alkaloid Localization in D. myoporoides R. Br. Cultured Tissues and Organs

F I G . 7. Fresh section from 14-week-old calli induced with BA 10 mM IBA 0.1 mM. The presence of stained cell contents in the concentric ring indicates alkaloid localization (iodoplatinate). Bar 100 mm. cr, Concentric ring. F I G . 8. Alkaloid-free section from 14-week-old calli induced with BA 10 mM IBA 0.1 mM. The absence of stained cell contents in the concentric ring indicates no alkaloid localization (iodoplatinate). Bar 25 mm. cr, Concentric ring. F I G . 9. Cross-section from fresh cultured root regenerated from calli induced with BA 1 mM IBA 10 mM. Stained xylem cell contents indicate alkaloid localization (iodoplatinate). Bar 100 mm. xy, Xylem cells. F I G . 10. Alkaloid-free de-stained cross-section from root regenerated from calli induced with BA 1 mM IBA 10 mM showing no alkaloid localization in the xylem cells ( platinic chloride). Bar 25 mm. xy, Xylem cells.

Khanam et al.Alkaloid Localization in D. myoporoides R. Br. Cultured Tissues and Organs 751
Localization of alkaloids in the vascular region of the basal stem sections indicates that alkaloids can be biosynthesized in the non-rooted shoots. Localization of alkaloids in the basal stem sections of the cultured nonrooted D. myoporoides shoot has not yet been reported. However, Mothes and Remeike (1952) (cited in Missaleva et al., 1993) reported that hyoscyamine is converted to scopolamine in the stem of Datura innoxia. In our shoots, the presence of nicotine, hyoscyamine and scopolamine was conrmed by GC-MS. The presence of tropane alkaloids in the non-rooted Duboisia myoporoides shoots has not been reported before. However, Missaleva et al. (1993) reported the presence of tropane alkaloids in cultured non-rooted Datura innoxia shoots. Tropane alkaloids were localized in the vascular region where large cells in the secondary xylem were present. In some alkaloid-producing plant species, alkaloid transport is mediated by carrier proteins (Warner and Matile, 1985; Matern et al., 1986; Wink, 1987). Wink (1987) reported signicant alkaloid production when gene expression for alkaloid biosynthesis and transport (which is related to accumulation) took place at the same time. Guern et al. (1987) reported that gene expression for both alkaloid biosynthesis and transport takes place in the presence of a suitable storage site where accumulation without degradation occurs. Since large xylem cells are dead, biosynthesis of enzymes is not possible in these cells. It may be that gene expression for alkaloid biosynthesis and the carrier proteins takes place after formation of the large cells in the secondary xylem. Formation of alkaloids in the non-rooted shoots may also be related to other cells. Further investigation related to other cell dierentiation is therefore necessary. No alkaloids were localized in the basal stem sections of the non-rooted shoots dierentiated from calli induced with combinations (1), (3) and (7). However, a signicant amount of only nicotine was detected by GC-MS in these shoots. Kitamura et al. (1985) detected a small amount of nicotine in the cultured non-rooted Duboisia myoporoides shoot. Our study did not show the biosynthetic site for alkaloids in the non-rooted shoot. Since basal stem and leaves were analysed together by GC-MS it is not clear whether nicotine was present in the leaves or in the basal stem. In intact D. leichhardtii plants, tropane alkaloids are selectively transported from roots to aerial parts while pyridine alkaloids remain in the roots (Yamada and Endo, 1984). Our study indicates that after biosynthesis, tropane alkaloids are selectively transported to the basal stem for storage. Nicotine may be transported to the basal stem but the amount was not identiable by the colour reagents used. Localization of tropane alkaloids was related to the presence of large cells in the secondary xylem whereas the presence of nicotine was not related to such xylem dierentiation. It appears that formation of nicotine in regenerated non-rooted shoots does not require the same degree of dierentiation needed for the complete plant, whereas tropane alkaloid formation does. The presence of alkaloids in 14-week-old calli induced with combination (9) indicates that either alkaloid biosynthesis takes place in calli cells or alkaloids formed in the shoots are returned to the callus where they accumulate. Kitamura et al. (1996) reported the recycling of alkaloid from the shoots to the roots in D. myoporoides. Since only hyoscyamine was detected by GC-MS, it may be that the calli attached to the shoot were used for accumulation and storage of the alkaloids biosynthesized in the non-rooted shoots. Localization of alkaloids in the xylem cells of the 4-weekold roots indicates that xylem dierentiation takes place earlier in the cultured roots without aerial organs than in the non-rooted shoots. Earlier formation of xylem cells in the regenerated roots than in the regenerated shoots may account for the earlier presence of tropane alkaloids in the former tissue. The presence of higher amounts of alkaloids in the 4-week-old roots than in the 9-week-old non-rooted shoots indicates higher alkaloid biosynthetic ability of the roots. Cytokinin and auxin combinations (2) and (9) caused tropane alkaloid localization in cultured non-rooted shoots, whereas other combinations did not. In the complete plant, cytokinin and auxin biosynthesis takes place in the root and shoot cells, respectively (Matthysse and Scott, 1984). Auxin levels produced in the shoot are related to other plant growth regulators produced in the root (Tal et al., 1979). Shoot culture media used in our study were supplemented with cytokinin and auxin only. So the eect of roots in a normal system was absent in the non-rooted shoot. After long incubation periods, two cytokinin and auxin combinations produced dierentiation as in the parent plant. Perhaps the older regenerated shoots were able to supplement the plant growth regulators provided to produce the required dierentiation and thereby stimulate alkaloid biosynthesis as in the parent plant. The above view is consistent with comparison of the biosynthetic ability of seedlings and regenerated plants. Kitamura et al. (1985) reported that leaves of D. myoporoides seedlings contained the largest amounts of alkaloids and the main alkaloids were present throughout development. On the other hand, leaves of the plantlets regenerated from the callus induced in the MS basal medium supplemented with Kin 4.5 mM 2,4-D 0.0465 mM contained no or few alkaloids and the amounts of alkaloids were extremely low during early stages of development. Later, the alkaloid contents resembled the amounts found in the intact plant. Our results indicate that cell dierentiation in the cultured non-rooted shoots is related to cytokinin/auxin combinations used at the callus induction stage but not to the procedure of shoot regeneration from non-organogenic and organogenic calli. This model can be further tested by investigating cell dierentiation for stable tropane alkaloid biosynthesis in the cultured shoots using various combinations and concentrations of dierent plant growth regulators. Development of xylem dierentiation in calli may help to express stable tropane alkaloid biosynthesis without the need to regenerate dierentiated organs. That is, this nding may allow commercial tropane alkaloid production from the calli with the dierentiated xylem but not requiring organ development.

752 Khanam et al.Alkaloid Localization in D. myoporoides R. Br. Cultured Tissues and Organs
AC K N OW L E D G E M E N T S This report is based on the senior author's PhD Thesis, Relationship between organogenesis, dierentiation and histolocalization of selected alkaloids in Duboisia myoporoides R. Br. submitted to the University of Western Sydney, Macarthur, Australia, June 1999.
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We thank sta at the Mount Annan Botanic Gardens for supplying Duboisia myoporoides R. Br. plant material, Dr Philip Tong of AGAL for carrying out GC-MS analyses and Dr Mary Campbell for critical examination of this manuscript. The senior author acknowledges nancial support from her husband and UWS Macarthur during her PhD candidature.

L I T E R AT U R E C I T E D
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